Estrogen induces c-myc gene expression via an upstream enhancer activated by the estrogen receptor and the AP-1 transcription factor

c-myc oncogene is implicated in tumorigenesis of many cancers, including breast cancer. Although c-myc is a well-known estrogen-induced gene, its promoter has no estrogen-response element, and the underlying mechanism by which estrogen induces its expression remains obscure. Recent genome-wide studies by us and others suggested that distant elements may mediate estrogen induction of gene expression. In this study, we investigated the molecular mechanism by which estrogen induces c-myc expression with a focus on these distal elements. Estrogen rapidly induced c-myc expression in estrogen receptor (ER)-positive breast cancer cells. Although estrogen had little effect on c-myc proximal promoter activity, it did stimulate the activity of a luciferase reporter containing a distal 67-kb enhancer. Estrogen induction of this luciferase reporter was dependent upon both a half-estrogen response element and an activator protein 1 (AP-1) site within this enhancer, which are conserved across 11 different mammalian species. Small interfering RNA experiments and chromatin immunoprecipitation assays demonstrated the necessity of ER and AP-1 cross talk for estrogen to induce c-myc expression. TAM67, the AP-1 dominant negative, partially inhibited estrogen induction of c-myc expression and suppressed estrogen-induced cell cycle progression. Together, these results demonstrate a novel pathway of estrogen regulation of gene expression by cooperation between ER and AP-1 at the distal enhancer element and that AP-1 is involved in estrogen induction of the c-myc oncogene. These results solve the long-standing question in the field of endocrinology of how estrogen induces c-myc expression.     

Gene-specific patterns of coregulator requirements by estrogen receptor-α in breast cancer cells

Progesterone receptor (PgR) controls the menstrual cycle, pregnancy, embryonic development, and homeostasis, and it plays important roles in breast cancer development and progression. However, the requirement of coregulators for estrogen-induced expression of the PgR gene has not been fully explored. Here we used RNA interference to demonstrate dramatic differences in requirements of 10 different coregulators for estrogen-regulated expression of six different genes, including PgR and the well-studied TFF1 (or pS2) gene in MCF-7 breast cancer cells. Full estrogen-induced expression of TFF1 required all ten coregulators, but PgR induction required only four of the 10 coregulators. Chromatin immunoprecipitation studies demonstrated several mechanisms responsible for the differential coregulator requirements. Actin-binding coregulator Flightless-I, required for TFF1 expression and recruited to that gene by estrogen receptor-α (ERα), is not required for PgR expression and not recruited to that gene. Protein acetyltransferase tat-interactive protein 60 and ATP-dependent chromatin remodeler Brahma Related Gene 1 are recruited to both genes but are required only for TFF1 expression. Histone methyltransferase G9a is recruited to both genes and required for estrogen-induced expression of TFF1 but negatively regulates estrogen-induced expression of PgR. In contrast, histone methyltransferase myeloid/lymphoid or mixed-lineage leukemia 1 (MLL1), pioneer factor Forkhead box A1, and p160 coregulator steroid receptor coactivator-3 are required for expression of and are recruited to both genes. Depletion of MLL1 decreased ERα binding to the PgR and TFF1 genes. In contrast, depletion of G9a enhanced ERα binding to the PgR gene but had no effect on ERα binding to the TFF1 gene. These studies suggest that differential promoter architecture is responsible for promoter-specific mechanisms of gene regulation.